As it is known, Nerve Growth Factor (NGF) is a signaling protein involved in the development of the nervous system of vertebrates, which drives and regulates the growth and survival of neurons through cell signaling mechanisms. Such molecule is produced in many mammal tissues, including man, and is released in the bloodstream in higher amounts during the growth and differentiation of the nervous system. It is presently ascertained, actually, that NGF exerts a trophic, differentiative and tropic action on the cholinergic neurons of the central nervous system and of the peripheral nervous system, and is active more generally in the protection of the nervous system.
NGF was discovered in the 1950s by Rita Levi-Montalcini, who during thirty years continued her research on this protein molecule and the mechanism of action thereof. In 1986 she was awarded the Nobel Prize in medicine, together with the US researcher Stanley Cohen. Several studies by Montalcini and her group of researchers show that nerve cells grow when placed in contact with NGF in vitro, while the tropic action of NGF is shown by the directional growth of the nerve fibres of target cells towards an exogenous source of NGF. Thus, the production of such growth factor has the effect of causing survival of the target cells and steering the growth of their axons, so as to enhance the formation of correct synaptic contacts.
It is known that the biological effect of NGF is mediated by two receptors present on the surface of the corresponding target cells, i.e. the highaffinity TrkA (tyrosine kinase A) receptor and the low-affinity p75 receptor. The existence of many antibodies that selectively inhibit the biological effect of NGF has enabled an accurate characterisation and modulation of its activity, both in cellular systems and in vivo.
Several experimental works have shown the physiopathologic importance of NGF in preventing neuronal damage of a surgical, chemical, mechanical and ischemic origin, thus making it the ideal candidate for use not only in the prophylaxis but also in the therapy of many pathologies of the central and peripheral nervous system. For instance, NGF has been shown to be effective in the therapy of bedsores and chronic ulcers in general (Tuveri M. et al., NGF, a useful tool in the treatment of chronic vasculitic ulcers in rheumatoid arthritis, Lancet 2000, 356:1739-1740). In several cases patients with corneal neurotrophic ulcers, which arise in case of defective innervation, have been treated with eye-drops based on NGF obtaining a full restoration of the cornea (Bonini St. et al., Topical treatment with nerve growth factor for neurotrophic keratitis. Ophthalmology 2000, 107:1347-524; Lambiase A., et al., Anti-inflammatory and healing activities of nerve growth factor in immune corneal ulcers with stromal melting, Arch. Ophthalmol. 2000; 118:1446-9).
It has also been shown, more recently, that NGF eye-drops, upon administration on the corneal surface, can pass through the various ocular tissues, thus reaching internal structures of the eye, such as the retina and the optical nerve. Therefore, administration of NGF through the topic ophthalmic route has been proposed and successfully tested for the therapy of glaucoma as well, where such agent can perform a regeneration action on the optical nerve (Lambiase A. et al., Nerve growth factor eye drops to treat glaucoma, Drugs News Perspect. 2010, 23(6):361-7).
Taking into account the pathologies of the central nervous system, NGF has the property of nourishing the brain cells and preserving them from ageing and, in addition to deferring apoptosis of brain neurons, it increases their size and the important branchings connecting them with each other. This condition results in the so-called phenomenon of “neuronal plasticity”.
By using the mouse animal model, it has been experimentally shown that if the animals are deprived of NGF they have memory deficits similar to the deficits caused by Alzheimer's disease. Since the main factor responsible for Alzheimer's is a peptide, i.e. beta-amyloid, the production of which in normal cells is suppressed by NGF, if NGF is removed from a culture of neural cells the production of beta-amyloid is activated in a short time. NGF has also been reported to be capable of stopping the progression of atrophy of acetylcholine-producing neurons, the reduction of which is involved in the symptomatology of Alzheimer's disease (Auld D. S. et al., Alzheimer's disease and the basal forebrain cholinergic system: relations to β-amyloid peptides, cognition, and treatment strategies, Prog. Neurobiol. 2002, 68:209-245; Williams B. J. et al., Nerve growth factor in treatment and pathogenesis of Alzheimer's disease, Prog Neurobiol 2006, 80(3):114-28).
NGF is also released in the skin, not only by neuronal fibres but also by basal keratinocytes, mast cells (or mastocytes), Merkel cells. In particular, keratinocytes, the skin cells, also express both high affinity and low affinity NGF receptors. It has been shown that NGF, more than EGF (Epidermal Growth Factor), stimulates the keratinocytes prolipheration and prevents their apoptosis. Keratinocytes exert a protective action against external agents which may be noxious on the skin, attracting the arrival of immunocytes. Therefore, it is believed that NGF may play a role also in the protection from skin disorders such as sunburn, atopic dermatitis, T cells cutaneous lymphoma, scleroderma, paraneoplastic hyperproliferative lesions, psoriasis.
It is also known that mast cells, cells involved in the immune and inflammatory response, have in their interior hundreds of granules containing various substances. When the mast cell is called upon to counter a tissue damage, for instance of allergic or inflammatory origin, or due to infection or trauma, the mast cells degranulate, releasing the substances contained therein, and in this way they protect the surrounding tissue, thus promoting the recovery thereof. NGF is among the substances released by mast cells.
From the foregoing it is apparent that, irrespective of the cellular origin thereof, an increase of NGF in a lesion site may stimulate healing also through different mechanisms than the neuronal one. Indeed, the ability shown by NGF to influence the skin cells makes it an optimal candidate to the role of modulator of the healing phases, first of all the inflammatory phase. The induction and enhancement of mast cell degranulation is believed to be the fundamental mechanism through which NGF activates the inflammatory process. The proliferative action on keratinocytes explains the role of NGF in reepithelialization, which is the key event of cutaneous healing, through which keratinocytes migrate at the lesion margins and prolipherate until they completely cover the lesion.
It has been reported in the literature that the NGF concentration is high, in combination with a great increase of mast cells, in a number of inflammatory and autoimmune conditions, including experimental allergic encephalitis (EAE), the latter representing the most widely employed model for multiple sclerosis (Calzà et al., Proliferation and phenotype regulation in the subventricular zone during experimental allergic encephalomyelitis: In vivo evidence of a role for nerve growth factor, PNAS 1997, 95:3209-3214). Another example is represented by the studies carried out on allergic conjunctivis, where an increase of NGF due to inflammation conditions has been reported (Bonini St. et al., Nerve growth factor: an important molecule in allergic inflammation and tissue remodeling, Int. Arch. Allergy Immunol. 1999, 118:159-162).
Since it has been shown that Nerve Growth Factor plays an important role in protecting the neural cells, it would be extremely useful if NGF could be administered to patients suffering from pathologies of the nerve system, both central and peripheral (CNS and PNS), or to subjects which are exposed to the risk of developing such pathologies. However, in spite of the fact that NGF has been discovered more than six decades ago, a sufficiently cheap way of producing it has not been found so far. The synthesis of human NGF through genetic engineering techniques appears to be an extremely difficult and complex task, and for this reason the murine version of NGF has been mostly employed to date, for experimental and clinical purposes. Murine NGF is extracted from rat salivary glands, and has an extremely high extraction cost as well.
In addition, in the specific case of pathologies of the CNS, such as Alzheimer's disease or Parkinson's disease, an administration of NGF by the oral or the parenteral routes is hindered by the presence of the blood-brain barrier. The latter prevents the brain tissues from being reached by concentrations of active substances sufficient to exert an appreciable therapeutic action. Actually, all of the studies carried out so far have shown an effectiveness of NGF only when it is administered by the intracerebral route (intracerebroventricularly), since this molecule is unable to pass through the blood-brain barrier in therapeutic concentrations through systemic administration.
The literature also describes attempts to stimulate the endogenous production of NGF directly in the central nervous system, with the purpose of treating neurodegenerative disorders such as Alzheimer's by administration of a suitable chemical agent, with the role of “NGF inducer”. To this regard, the U.S. Pat. No. 5,281,607 (Stone et al., assigned to New York University) proposes the use of pharmacological agents, specifically α adrenergic receptor antagonists (a adrenergic antagonists or alpha-blockers) or β adrenergic receptor antagonists (beta-blockers), as agents stimulating the endogenous production of NGF. Such agents, among which the α2 receptor antagonists are preferred, are administered by the oral or parenteral route, although the intranasal route is mentioned among the possible routes of administration.
The U.S. Pat. No. 6,410,046 to E. Lerner (assigned to Intrabrain International) proposes a method for administering any medicament to the central nervous system through the olfactory zone, using the application of an electrical potential to obtain the transfer of the medicament through the nasal mucous membrane, in combination with the use of a permeation enhancer. The two agents, defined physical agent and chemical agent, can be applied sequentially or simultaneously to obtain the passage of the medicament to the brain through the intranasal or the transocular route. However, the introduction of an electrode in the nasal cavity and the use of an electric potential appear to be traumatic for the patient, besides being risky in view of the possible complications.
Compounds having similar pharmaceutical activity as the compounds proposed by Stone et al. are also used as “NGF inducers” in the US patent application US 2008/0014152 by M. Di Mauro et al., in particular clenbuterol, a β2 adrenergic receptor antagonist. Di Mauro uses an intranasal administration method similar to the method proposed by Lerner, replacing the electrical means with a mechanical means, namely a spray nasal dispenser of a commercial kind with a long thin semiflexible tube connected thereto, to be placed in the nasal cavity below the cribriform plate, the thin bone layer which separates the brain from the nose. According to the document, the beta blocking agent, being sprayed through the tube oriented with its distal exit hole close to the cribriform plate, directly reaches the brain, where it exerts its action of inducing the production of endogenous NGF.
Another patent document of the same main author, the patent application US 2007/0031241 (M. Di Mauro et al.), discloses a device for easing the passage of specific medicinal substances through the cribriform plate or the meninges. The system disclosed is based, similarly to Lerner's proposal, on applying a voltage to administer therapeutical agents to the central nervous system by the intranasal route, across the cribriform plate. The administration is made by introducing a delivery device, for instance a cathodic probe, into the nasal cavity. The delivery device may have a similar shape as the devices proposed in the two previously cited documents, so as to be placed close to the cribriform plate. In this case the medicinal substances involved in the iontophoretic transfer are substances having antioxidant activity, suitable to reduce oxidative stress in the brain tissues, and not NGF inducers.
It is to be noted that the intranasal administration of pharmacologically active substances in order to obtain a bioavailability of the concerned substances in the CNS involves the actual risk of serious complications; it is dangerous since it does not take into account the fact that the cribriform plate has a distance from the nasal vestibule which varies from person to person. In addition, the bone thickness is variable, and in many cases such bone is extremely thin, so that the patient may be exposed to the risk of breaking the cribriform plate itself and to the risk of a resulting meningitis, which is a potentially lethal occurrence.
Summarizing, the prior art solutions intended to stimulate the endogenous production of NGF, or which could be used to that aim, show various drawbacks, including:                the known difficulties due to overcoming the blood-brain barrier, it the route of administration considered is the oral or the parenteral route;        the risk due to possible noxious effects of exogenous chemical agents, such as alfa-blockers or beta-blockers, when introduced directly in the brain tissues;        the risk due to the use of electrical stimuli in the nose of in the brain;        the mechanical risk due to the introduction of a delivery device, of any nature, in close proximity to the cribriform plate.        
Therefore, there was a need in the prior art to find solutions which would be free from the drawbacks mentioned above.
In the frame of the research that led to the present invention, the complex series of experimental findings which led to consider nerve growth factor as a mediator of the inflammatory process. As it is known, inflammation is a response to a tissue damage having the object of insulating the zone damaged by the harmful agents and preventing the latter from diffusing into the body, and of attracting in that zone cells having anti-infective activity (leucocytes) and, finally, stimulating healing.
A fundamental role is also played by mast cells (or mastocytes), which are normally present in the connective tissue. The latter tend to concentrate particularly along the blood vessels and are now considered to be the activators of acute phlogosis. In response to an inflammatory stimulus, mast cells play a key role in starting inflammation, as they put into action substances already stored in their cytoplasmic granules, which are immediately released, and newly synthesized substances, or substances which attract (chemotaxis) white blood cells in the inflammatory zone. White blood cells play a defensive role against infection and produce themselves inflammatory mediators.
Several endogenous compounds play an ascertained role as inflammatory mediators. These include, firstly, preformed mediators, released from the mast cells secretion granules, from the secretion granules of granulocytes (which belong to the white blood cells group) and from the platelets in the extracellular environment, such as histamine and serotonin, and secondly ex novo synthesized mediators, which are synthesized in case of need, such as prostaglandins, leukotrienes, platelet activating factors (PAF), reactive oxygen species (ROS), nitrogen oxide (NO), cytokines.
The substances known as inflammatory mediators, in that they are produced by cells which take part in the inflammatory mechanism, or by cells active in attracting leukocytes (i.e. white blood cells) and mast cells in the inflammatory zone, include, as mentioned above, nerve growth factor (NGF) and the other two neurotrophins known as neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4). In addition, inflammatory mediators include heparin and the other proteoglycans, serotonin, eosinophil chemotactic factor of anaphilaxis (ECF-A) and substance P.
As already noted, NGF, produced by the mast cells and released from their granules, but also produced by eosinophil granulocytes and by macrophages—all these cells taking part in the inflammatory process—is a chief molecule of a complex nerurotrophin family, which comprises neurotrophin 3 (NT-3) and neurotrophin 4 (NT-4). The latter have a protective activity on the nervous system cells which is similar to the action of NGF (Francis N. et al., NT-3, like NGF, Is Required for Survival of Sympathetic Neurons, but Not Their Precursors, Dev. Biol. 1999, 210: 411-423; Ulupinar E. et al., Differential Effects of NGF and NT-3 on Embryonic Trigeminal Axon Growth Patterns, J. Comp. Neurol. 2000, 425:201-218; Cheryl L. et al, Neurotrophin-4: A Survival Factor for Adult Sensory Neurons, Curr. Biol. 2002, 12:1401-1404).
Serotonin (or 5-hydroxytryptamine, 5-HT) is a neurotransmitter synthesized in the serotoninergic neurons of the CNS and in the enterochromaffin cells of the gastrointestinal tract, which is mainly involved in the regulation of mood. As noted, serotonin is considered to be a preformed inflammatory mediator, as it is present in the dense granules of platelets. The serotonin receptors are found mainly on the cell membrane of neurons in the central and peripheral nervous system, where serotonin performs its action. It plays an important role in appetite control and in the alimentary behaviour, on headache, on blood pressure, on premature ejaculation and on sleep.
Serotonin promotes the appearance of satiety feeling and in general the reduction of ingested food. For this reason some anorexia-inducing drugs, such as fenfluramine, act by increasing the serotonin signal. In addition, serotonin stimulates the reduction of carbohydrates intake due to a negative feedback. Actually, the ingestion of carbohydrates stimulates insulin production, which eases the entrance of nutrients, including amino acids and with the exception of tryptophan, in cells. Thus, the relative blood levels of tryptophan increase, this increase enhancing its entrance in the central nervous system, where it stimulates serotonin production.
Serotonin shows a euphorising and calming effect, and it is known that a correct amount of serotonin reduces stress, induces sleep and regulates the sleep cycles. It has been shown, inter alia, that the pharmacological treatment with serotonin enhances the quantity and quality of sleep. At the CNS level, serotonin is released from the presynaptic axon terminal, a part thereof acts on the postsynaptic receptor and the exceeding part is reabsorbed by the presynaptic terminal and stored in vesicles, or it is degraded by the monoamino oxidases (MAO). The MAO-inhibitor drugs act by blocking monoamino oxidases, thus bringing about in the CNS an increase of serotonin and of other brain monoamines. Well-known psychiatric medications, such as SSRIs (Selective Serotonin Reuptake Inhibitors), tricyclic antidepressants and MAO inhibitors are active on this neurotransmitter.
Some drugs with antiemetic properties are agonists of the serotonin receptors, with the aim of thus increasing its signal.
From the foregoing it is clear that a treatment capable of increasing the endogenous production of serotonin would meet a great interest in therapy.
Substance P (SP) is a short chain polypeptide that functions as a neurotransmitter and as a neuromodulator in mammals. It is produced and released by eosinophils, macrophages, endothelial cells, nerve terminals. An important role in the stimulation of SP synthesis is played by mast cells, which, upon an irritative stimulus, release endocellular inflammatory mediators and attract eosinophils and macrophages in the inflamed zone.
It is known that substance P plays an important role in pain perception and in modulation of vomiting, and is a potent vasodilator. It is also believed that the release of this neurotransmitter from the peripheral terminals of the sensory nerve fibres is involved in neurogenic inflammation, which in turn appears to be involved in the pathogenesis of headache. Actually, high concentrations of substance P prevent or reduce headache.
Another inflammatory mediator which is released by the mastocyte granules is heparin, a glycosaminoglycan having a natural anticoagulant activity. Heparin is used as a blood fluidifyer, especially in patients with a greater tendency to blood coagulation or risk factors such as atrial fibrillation, deep venous thrombosis, in subjects undergoing dialysis, to prevent the formation of thrombi and the risk of complications, also lethal, such as pulmonary thrombosis or myocardial infarction. Heparin may play a role in the prevention of clot formation in patients who underwent heart surgery or coronary stent placement.
A further inflammatory mediator of interest herein is the eosinophil chemotactic factor of anaphylaxis, ECF-A, which is mainly produced by mast cells, and has the function of attracting eosinophil granulocytes in the inflammation zone. Eosinophils represent one of the three types of granulocytes present in the blood (i.e., neutrophils, basophils and eosinophils), which are active mainly in the immune response against parasites and in allergic responses.